Cannabinoids are a class of specialized compounds synthesized by Cannabis. They are formed by condensation of terpene and phenol precursors. As shown in FIG. 1, they include these more abundant forms: Delta-9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC), and cannabigerol (CBG). Another cannabinoid, cannabinol (CBN), is formed from THC as a degradation product and can be detected in some plant strains.
Typically, THC, CBD, CBC, and CBG occur together in different ratios in the various plant strains. These cannabinoids are generally lipophilic, nitrogen-free, mostly phenolic compounds and are derived biogenetically from a monoterpene and phenol, the acid cannabinoids from a monoterpene and phenol carboxylic acid, and have a C21 base. Cannabinoids also find their corresponding carboxylic acids in plant products. In general, the carboxylic acids have the function of a biosynthetic precursor. For example, these compounds arise in vivo from the THC carboxylic acids by decarboxylation the tetrahydrocannabinols Δ9- and Δ8-THC and CBD from the associated cannabidiol.
Cannabinoids are classified into two types, neutral cannabinoids and cannabinoid acids, based on whether they contain a carboxyl group or not. It is known that, in fresh plants, the concentrations of neutral cannabinoids are much lower than those of cannabinoid acids. Thus, as generally shown in FIG. 2, THC and CBD may be derived artificially from their acidic precursor compounds tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA) by non-enzymatic decarboxylation.
As detailed below, efforts to generate Cannabis plants that produce and/or accumulate high levels of cannabinoids have raised a number of technical problems. Chief among them is the fact that cannabinoid synthesis produces toxic by-products. Notably, both CBDA and THCA synthases require molecular oxygen, in conjunction with a molecule of FAD, to oxidize Cannabigerolic acid (CBGA). Specifically, as shown in FIG. 3, two electrons from the substrate are accepted by an enzyme-bound FAD, and then transferred to molecular oxygen to re-oxidize FAD. CBDA and THCA are synthesized from the ionic intermediates via stereoselective cyclization by the enzymes. As also noted in FIG. 3, the hydride ion is transferred from the reduced flavin to molecular oxygen, resulting in the formation of hydrogen peroxide (H2O2) and re-activation of the flavin for the next cycle. As a result, in addition to producing CBDA and THCA respectively, this reaction produces hydrogen peroxide which is naturally toxic to the host cell. Due to this production of a toxic hydrogen peroxide byproduct, cannabinoid synthesis generates a self-limiting feed-back loop preventing high-level production and/or accumulation of cannabinoids in in vivo systems.
Cannabis plants deal with these cellular cytotoxic effects is through a process of directing cannabinoid production to extracellular structures. Specifically, cannabinoid biosynthesis is localized in the secretory cavity of the glandular trichomes which are abundant on the surface of the female inflorescence in Cannabis sativa. Trichomes can be visualized as small hairs or other outgrowths from the epidermis of a Cannabis plant. For example, Tetrahydrocannabinolic acid (THCA) synthase is a water-soluble enzyme that is responsible for the production of THC. For example, as generally shown in FIG. 4, THC biosynthesis occurs in glandular trichomes and begins with condensation of geranyl pyrophosphate with olivetolic acid to produce cannabigerolic acid (CBGA); the reaction is catalyzed by an enzyme called geranylpyrophosphate:olivatolate geranyltransferase. CBGA then undergoes oxidative cyclization to generate tetrahydrocannabinolic acid (THCA) in the presence of THCA synthase. THCA is then transformed into THC by non-enzymatic decarboxylation. Sub-cellular localization studies using RT-PCR and enzymatic activity analyses demonstrate that THCA synthase is expressed in the secretory cells of glandular trichomes, and then is translocated into the secretory cavity where the end product THCA accumulates. THCA synthase present in the secretory cavity is functional, indicating that the storage cavity is the site for THCA biosynthesis and storage. In this way, the Cannabis is able to produce cannabinoids extracellularly and thereby avoid the cytotoxic effects of these compounds. In addition to cannabinoids, trichomes in Cannabis are also the sites of production of other secondary compounds like terpenes, which are responsible for the distinctive aroma of Cannabis. As such, the ability of Cannabis plants to produce and accumulate cannabinoids and terpenes is limited by the number and size of the plant's trichomes. As a result, the ability of Cannabis plant to produce and accumulate cannabinoids and terpenes is limited by the number of trichomes that are present. Increasing trichome density will increase production of cannabinoids and terpenes in Cannabis. 
Efforts to generate Cannabis strains that produce or accumulate high levels of cannabinoids have raised a number of technical problems. Chief among them is the fact that cannabinoid synthesis produces toxic by-products. Notably, both CBDA and THCA synthases require molecular oxygen, in conjunction with a molecule of FAD, to oxidize Cannabigerolic acid (CBGA). Specifically, as shown in FIG. 3, two electrons from the substrate are accepted by an enzyme-bound FAD, and then transferred to molecular oxygen to re-oxidize FAD. CBDA and THCA are synthesized from the ionic intermediates via stereoselective cyclization by the enzymes. The hydride ion is transferred from the reduced flavin to molecular oxygen, resulting in the formation of hydrogen peroxide and re-activation of the flavin for the next cycle. As a result, in addition to producing CBDA and THCA respectively, this reaction produces hydrogen peroxide (H2O2) which is naturally toxic to the host cell. Due to this production of a toxic hydrogen peroxide byproduct, cannabinoid synthesis generates a self-limiting feed-back loop preventing high-level production and/or accumulation of cannabinoids in in vivo systems. One way that Cannabis plants deal with these cellular cytotoxic effects is through the use of trichomes for Cannabinoid production and accumulations.
As will be discussed in more detail below, the current inventive technology overcomes the limitations of cannabinoid production systems while meeting the objectives of a truly effective high-level cannabinoid production and accumulation system.